Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction

Kirisawa, Rikio; Endo, Akifumi; Iwai, Hiroshi; Kawakami, Yoshimi

doi:10.1016/0378-1135(93)90128-t

Cited by 76 publications

(51 citation statements)

References 15 publications

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“…EHV-1 causes acute upper respiratory disease, central nervous system disorders and contagious virus abortion in horses, and EHV-4 causes respiratory disease and, occasionally, abortion [1]. The difference between EHV-1 and -4 has been demonstrated in pathogenicity in baby mice [11][12][13], restriction endonuclease analysis of viral DNA [18], and polymerase chain reactions [2,6,15]. However, because of the extensive antigenic cross-reactivity, it is difficult to distinguish between EHV-1 and -4 infections by conventional serological tests.…”

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confidence: 99%

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Diagnosis and Sero-Epizootiology of Equine Herpesvirus Type 1 and Type 4 Infections in Japan Using a Type-Specific ELISA.

et al. 1998

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ABSTRACT. Recently, a type-specific ELISA using equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) glycoprotein Gs (gGs) was developed by Crabb and Studdert [1993]. To investigate the dissemination of EHV-1 and -4 among horses in Japan, we applied their ELISA as suitable for discriminating between EHV-1 and -4 infections serologically. Type-specificity of the ELISA was confirmed by using paired sera of infected horses with either EHV-1 or -4. Application of the ELISA to sera collected before and after the winter season of 1995-1996 from 80 racehorses revealed that 30 horses showed significant antibody responses against EHV-1 and 9 against EHV-4, respectively. The results indicated that this ELISA using paired sera is useful for a diagnosis and an epizootiological study on EHV-1 and -4 infections. -KEY WORDS: diagnosis, epizootiology, equine herpesvirus.J. Vet. Med. Sci. 60(10): 1133-1137, 1998 Virological diagnosis can identify the type of infected virus, but they are time-consuming and laborious. Therefore, it is considered to be very important to diagnose the type of infected virus serologically. The purpose of this study was to develop a type-specific serological test for diagnosis of EHV-1 and -4 infections, which facilitates the elucidation of epizootiological characteristics of both infections among the various horse populations in Japan.The EHV-1 89c25p strain [9] and an EHV-4 strain TH20, the Japanese prototype of EHV-4 [4], were used in this study. Viruses were propagated at low multiplicities of infection in primary fetal horse kidney cells. Infected cells showing a cytopathic effect were treated with 1% sodium dodecyl sulfate (SDS) and proteinase K (0.1 mg/ml) in 0.1 M Tris-HCl (pH 8.0), 0.1M NaCl, 5 mM EDTA at 37°C overnight. Following the DNA extraction with phenol and chloroform-isoamyl alcohol (24:1), ethanol precipitated preparations were dissolved in water.Crabb and Studdert [3] reported that type-specific, continuous epitopes were located in the corresponding Cterminal variable region of EHV-1 gG (amino acids 288 to 350) and EHV-4 gG (amino acids 287 to 382), which show little amino acid identity. To express the type-specific antigen, the bacterial expression plasmid pGEX-5X1 was used. The recombinant pGEX-5X1 plasmids, pGEX-gG1P and pGEX-gG4P, were constructed according to the methods described previously [3] with a minor modification. (i) pGEX-gG1P was constructed by PCR amplification (35 cycles of 92°C for 1 min, 55°C for 1 min, 72°C for 1 min) with the EHV-1 89c25p DNA as the template and the following primers: forward primer, gG1P-F, containing an EcoRI site, 5'-CCGAATTCGGTGACGAAACATACGA-3', and reverse primer, gG1P-R, containing an XhoI site, 5'-AACTCGAGCTGGATGCCGTTCGACG-3'. The amplified fragment was digested with EcoRI and XhoI and cloned into the pGEX-5X1 digested with the same restriction endonucleases. (ii) pGEX-gG4P was constructed by PCR amplification (35 cycles of 92°C for 1 min, 50°C for 1 min,

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confidence: 99%

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] in which a tentative serological diagnosis of EHV-1 or -4 infection was made by complement fixation (CF) test. The result showed that a final diagnosis of EHV-1 infection was made in 8 horses and that diagnosis of EHV-1 infection might be made in another 3 horses (Table 1).…”

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confidence: 99%

Diagnosis and Sero-Epizootiology of Equine Herpesvirus Type 1 and Type 4 Infections in Japan Using a Type-Specific ELISA.

et al. 1998

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“…The primer sequences were selected based on results obtained by Kirisawa et al (1993). The 20 base biotinylated (B) forward primer sequence (P1) was compatible with both EHV-1 and EHV-4 while the reverse 20 base sequence (P2) was compatible with only EHV-1.…”

Section: Ehv-1 Oligonucleotide Primersmentioning

confidence: 99%

“…The development of the polymerase chain reaction (PCR) has lead to improved detection of EHV-1 DNA from clinical and cell culture material (Kirisawa et al, 1993). Amplification of viral DNA by PCR can be further enhanced by hybridisation with viral specific DNA probes (Lawrence et al, 1994;Welch et al, 1992;Kirisawa et al, 1993) and also by restriction endonuclease digestion (Allen et al, 1983;Morris and Field, 1988;Palfi and Christensen, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Amplification of viral DNA by PCR can be further enhanced by hybridisation with viral specific DNA probes (Lawrence et al, 1994;Welch et al, 1992;Kirisawa et al, 1993) and also by restriction endonuclease digestion (Allen et al, 1983;Morris and Field, 1988;Palfi and Christensen, 1995). However, the extraction of viral DNA from samples may also lead to the co-purification of PCR inhibitors (Zerbini et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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The development of a competitive PCR–ELISA for the detection of equine herpesvirus-1

Daly

Doyle

2003

Journal of Virological Methods

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Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR Á/ELISA system. A competitive PCR Á/ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR Á/ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms. #

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First molecular detection of Equine Herpesvirus type 3 (EHV‐3) in Chile

Troncoso

Calvanese

Saravia

et al. 2022

Veterinary Medicine & Sci

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Equine coital rash (ECE) is a highly contagious benign infection that induces lesions on external genitals, and it is caused by the equine herpesvirus type 3 (EHV-3). Although the disease is globally distributed, its presence in Chile has not been documented from a genetic point of view. Here, we performed polymerase chain reaction screenings for EHV-3 in lesions of external genitals in four horses belonging to a riding station at Bulnes, Ñuble Region, Chile. We sequenced a fragment of the glycoprotein G (gG) gene from three horses with clinical signs of ECE. The sequences were identical between them and 99.7% similar to a haplotype of EHV-3 detected in Brazil, and phylogenetically related with homologue from Japan, Russia and Brazil. Our results show the presence of EHV-3 for the first time in horses with ECE in Chile.

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Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction

Cited by 76 publications

References 15 publications

Diagnosis and Sero-Epizootiology of Equine Herpesvirus Type 1 and Type 4 Infections in Japan Using a Type-Specific ELISA.

Diagnosis and Sero-Epizootiology of Equine Herpesvirus Type 1 and Type 4 Infections in Japan Using a Type-Specific ELISA.

The development of a competitive PCR–ELISA for the detection of equine herpesvirus-1

First molecular detection of Equine Herpesvirus type 3 (EHV‐3) in Chile

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